DNA biochip and methods of use

ABSTRACT

The subject invention concerns materials and methods for detecting nucleic acid sequences. One aspect of the invention concerns a silicon-based “biochip” comprising nucleic acid immobilized thereon. In one embodiment, the silicon comprises microcavities. The nucleic acid to be assayed for the presence of one or more target nucleic acid sequences is immobilized on the silicon. A nucleic acid, such as an oligonucleotide probe, having a sequence substantially complementary to the target nucleic acid sequence can be used to detect the immobilized nucleic acid on the silicon. If the nucleic acid used for detection hybridizes with a target nucleic acid sequence, the hybridized sequences can be detected directly or indirectly. In an exemplified embodiment, the oligonucleotide probe can be labeled with a detectable label, for example, a fluorescent molecule. The subject invention also concerns methods for detecting a target nucleic acid using a silicon-based biochip of the invention.

CROSS-REFERENCE TO A RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/347,438, filed Feb. 3, 2006, now U.S. Pat. No. 7,875,426, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/649,961,filed Feb. 4, 2005, each of which is hereby incorporated by referenceherein in its entirety, including any figures, tables, and drawings.

BACKGROUND OF THE INVENTION

DNA plays an important role in many cellular processes like replication,homologous recombination and transcription. Besides its genomicinformation, DNA exhibits very interesting biophysical andphysicochemical properties which are essential for proper functioning ofthe biomolecular processes involved. Biochips, particularly those basedon DNA are powerful devices that integrate the specificity andselectivity of biological molecules with electronic control and parallelprocessing of information. This combination will potentially increasethe speed and reliability of biological analysis. Microelectronictechnology is especially suited for this purpose since it enableslow-temperature processing and thus allows fabrication of electronicsdevices on a wide variety of substances like glass, plastic, stainlesssteel and silica wafer. Fundamental phenomena like molecular elasticity,binding to protein, supercoilling and electronic conductivity alsodepends on the numerous possible DNA confirmations and can beinvestigated nowadays on a single molecule level. Experiments withsingle DNA have been reported with scanning tunneling microscopy(Guckenberger et al., 1994), fluorescence microscopy (Yanagida et al.,1983), fluorescence correlation spectroscopy (Wannmalm et al., 1997),optical tweezers (Smith et al., 1996), bead techniques in magneticfields (Wang et al., 1997), optical microfibers (Strick et al., 1996),electron holography (Smith et al., 1992a) and atomic force microscopy(Cluzel et al., 1996; Fink et al., 1999; Hansma et al., 1991). All thesemethods provide, directly or indirectly, information on molecularstructure and function. They differ, however, in the molecularproperties they probe, their spatial and temporal resolution, theirmolecular sensitivity and working environment.

Fluorescently labeled oligonucleotide probes are in regular use fornucleic acid sequencing (Mirzabekov, 1994), sequencing by hybridization(SBH) (Speel et al., 1999), fluorescence in situ hybridization (FISH)(Lakowicz et al., 1999), fluorescence resonance energy transfer (FRET)(Selvein, 2000), molecular beacons (Singh et al., 2000), taqman probes(Broude, 2002), and chip-based DNA arrays (Wittwer et al., 1997). Thishas made fluorescent probes an important tool for clinical diagnosticsand made possible real-time monitoring of oligonucleotide hybridization.Furthermore, fluorescent-based diagnostics avoid the problem of storage,stability, and disposal of radioactive labels (Schena, 2000; Drobyshovet al., 1997).

Knowledge of structural and physical properties in microbial cells andmicrobial cell components is required to obtain a comprehensiveunderstanding of cellular process and their dynamics. The need for anondestructive method was satisfied with the development of the AtomicForce Microscope (AFM). The last 15 years have witnessed theextraordinary growth of structural studies in biology, and the impact isbeing felt in almost all areas of biological research. Several groupshave used microscopy for the analysis of DNA, protein, and DNA-proteininteractions. Until recently, electron microscopy was used as the maintool for imaging DNA; however this technique can be harsh on biologicalsamples, making successful analysis extremely difficult. Approximately adecade ago, scientists began to use AFM for the analysis of biologicalsamples. AFM allowed the analysis of biological molecules to beperformed faster, easier and more accurately yielding successfulcharacterization of biological specimens. The development of the AFM andits introduction for imaging biological samples has provided scientistswith a very powerful tool to explore many aspects of protein-protein,protein-DNA and many other interactions (Fritz et al., 2000).

Various methods can be employed to bind DNA to different hosts. An arrayof substances, including catalytic antibodies, DNA, RNA, antigens, livebacterial, fungal, plant and animal cells, and whole protozoa, have beenencapsulated in silica, organosiloxane and hybrid sol-gel materials.Sol-gel immobilization leads to the formation of advanced materials thatretain highly specific and efficient functionality of the guestbiomolecules within the stable host sol-gel matrix (Hench et al., 1990).The protective action of the sol-gel cage prevents leaching andsignificantly enhances stability of biomolecules within the sol-gel. Theadvantages of these ‘living ceramics’ might give them applications asoptical and electrochemical sensors, diagnostic devices, catalysts, andeven bio-artificial organs. With rapid advances in sol-gel precursors,nanoengineered polymers, encapsulation protocols and fabricationmethods, this technology promises to revolutionize bioimmobilization.Biosensors using immobilized receptors are finding ever-increasingapplication in a wide variety of fields such as clinical diagnostics,environmental monitoring, food and drinking water safety, and monitoringof illicit drugs (Brinker et al., 1985). One of the most challengingaspects in development of these sensors is immobilization andintegration of biological molecules in the sensor platform. Numeroustechniques, including physical covalent attachment, and entrapment inpolymer and inorganic matrices, have been explored over the past decade.Sol-gel processes are promising host matrices for encapsulation ofbiomolecules such as enzymes, antibodies, and cells (Kumar et al.,2000).

Porous silicon (PS) was discovered in 1956 by Uhlir (Uhlir, 1956) whileperforming electropolishing experiments on Silicon wafers using ahydrofluoric acid (HF)-containing electrolyte. Uhlir found that byincreasing the current over a certain threshold, a partial dissolutionof the silicon wafer started to occur. Porous Silicon formation can beobtained by electrochemical dissolution of Silicon wafers in aqueous orethanoic HF solutions.

Microcavities are of interest for a wide range of fundamental andapplied studies, including investigations of cavity quantumelectrodynamics (Smith et al., 1992b), optical elements fortelecommunications (Goryachev et al., 2003), single-photon sources (Chanet al., 2000), and chemical or biological sensors (Isola et al., 1998).Microfabrication techniques allow reproducible fabrication of resonatorswith lithographically controlled dimensions. Using a combination oflithography and etching, semiconductor microcavities have been obtained.

Almost all children under two years of age are infected by RSV. Childrenwith weaker immune systems are at greater risk. For better health of allinfants, infants with symptoms of common cold, wheezing, pneumonia andbronchiolitis need to be diagnosed for the RSV infection. All hospitalsand physicians providing pediatric health care need RSV diagnosis kits.Current methods of detection are based on one single technology, i.e.,immunological assays and they are very expensive and have lowsensitivity and specificity. A new more robust technology is needed todiagnose children infected with RSV with higher sensitivity andspecificity and at a very lower cost.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods for detecting atarget nucleic acid comprising a nucleotide sequence of interest. Oneaspect of the invention concerns a silicon-based “biochip” comprisingnucleic acid immobilized thereon. In one embodiment, the silicon-basedbiochip comprises microcavities. The nucleic acid to be assayed for thepresence of one or more target nucleic acid sequences is immobilized onthe silicon. In one embodiment, the nucleic acid is provided in asol-gel composition. The nucleic acid can be immobilized in singlestranded form. A detector nucleic acid, such as an oligonucleotideprobe, having a sequence substantially complementary to the targetnucleic acid sequence can be used to detect the immobilized nucleic acidon the silicon. If the nucleic acid used for detection hybridizes with anucleotide sequence of a nucleic acid immobilized on the silicon, thehybridized sequences can be detected by direct or indirect means andthus the target nucleic acid is thereby detected. In an exemplifiedembodiment, the oligonucleotide probe can be labeled with a detectablelabel, for example, a fluorescent molecule.

The subject invention also concerns methods for detecting a targetnucleic acid using a silicon-based biochip of the invention. In oneembodiment, a sample to be tested for the presence of a target nucleicacid is contacted with a silicon biochip of the invention such thatnucleic acid in the sample is immobilized on the silicon biochip.Preferably, the silicon is prepared so as to have microcavities. Thenucleic acid to be assayed for the presence of one or more targetnucleic acid sequences can be provided on the silicon surface in asol-gel. The silicon biochip is then contacted with a detector nucleicacid that comprises a nucleotide sequence that is substantiallycomplementary with the sequence of the target nucleic acid of interestunder conditions that permit hybridization of the detector nucleic acidto the target nucleic acid. In one embodiment, the detector nucleic acidis labeled with a detectable moiety, such as a fluorescent molecule.Hybridization of the detector nucleic acid is indicative of the presenceof the target nucleic acid. The present methods can be used to detectnucleic acid sequences associated with bacteria, viruses, fungi,protozoans, and the like. In an exemplified embodiment, the targetnucleic acid sequence is from Respiratory Syncytial Virus (RSV).

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with the color drawings will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 shows the schematics of electrochemical etching of a siliconwafer.

FIGS. 2A-2C show the preparation of DNA fixation and hybridization withfluorescent molecules on porous silicon (PS) using TEOS.

FIG. 3 shows an SEM picture of porous silicon.

FIGS. 4A-4C show images of porous silicon with microcavities throughOptical Microscopic investigation of DNA biochip; FIG. 4A (10×), FIG. 4B(40×) and FIG. 4C (100×).

FIGS. 5A-5C show porous silicon microcavities attached with sDNA throughOptical Microscopic investigation of DNA biochip; FIG. 5A (10×), FIG. 5B(40×) and FIG. 5C (100×).

FIGS. 6A-6C show DNA hybridization with fluorescence attached cDNAmolecule with sDNA through Optical Microscopic investigation of DNAbiochip; FIG. 6A (10×), FIG. 6B (40×) and FIG. 6C (100×).

FIG. 7A shows single stranded DNA attached to microcavity and FIG. 7Bshows a magnified view of single stranded DNA attached to microcavity.

FIG. 8A shows a 3D AFM picture of an sDNA bundle attached to microcavityon silicon wafer and FIG. 8B shows an AFM Images analysis of DNAhybridization (interaction of sDNA with cDNA.

FIG. 9 shows PL spectra of: sample “a” and “b” (sDNA on porous silicon),and PL spectra of sample “c” and “d” (cDNA hybridized to sDNA on poroussilicon).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is an oligonucleotide sequence corresponding to a sequencein the genome of Respiratory Syncytial Virus (RSV) F that is used in anexemplified embodiment of the present invention.

SEQ ID NO: 2 is an oligonucleotide sequence that is complementary to thesequence of SEQ ID NO: 1 and that is used in an exemplified embodimentof the present invention.

SEQ ID NO: 3 is the genomic nucleotide sequence for a RespiratorySyncytial Virus.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns materials and methods for detecting atarget nucleic acid. One aspect of the invention concerns asilicon-based “biochip” comprising nucleic acid immobilized thereon. Inone embodiment, the silicon comprises microcavities. Nucleic acid thatis to be assayed for the presence of one or more target nucleic acidsequences is immobilized on the silicon. In one embodiment, the nucleicacid is immobilized in single stranded form. In a further embodiment,the nucleic acid is immobilized on the silicon using a sol-gelcomposition.

Sol-gel compositions and methods for incorporating a biomolecule, suchas a nucleic acid, in sol-gel compositions are known in the art and havebeen described in U.S. Pat. Nos. 6,495,352 and 6,303,290, and in Kumaret al. (2000). A nucleic acid, such as an oligonucleotide probe, havinga nucleotide sequence substantially complementary to a target nucleicacid sequence can be used to detect the immobilized nucleic acid on thesilicon. If the nucleic acid used for detection hybridizes with a targetnucleic acid sequence, the hybridized sequences can be detected eitherby direct or indirect means. In an exemplified embodiment, a nucleicacid (e.g., an oligonucleotide probe) can be labeled with a detectablelabel, for example, a fluorescent molecule.

The subject invention also concerns methods for detecting a targetnucleic acid using a silicon-based biochip of the invention. In oneembodiment, a sample to be tested for the presence of a target nucleicacid is contacted with the surface of a silicon biochip of the presentinvention such that nucleic acid present in the sample binds to andbecomes immobilized on the silicon. Preferably, the silicon is preparedso as to have microcavities. The nucleic acid containing sample to beassayed for the presence of one or more target nucleic acid sequencescan be provided on the silicon surface in a sol-gel composition.Optionally, the biochip can be washed to remove unbound nucleic acid.The silicon biochip is then contacted with a detector nucleic acid thatcomprises a nucleotide sequence that is substantially complementary withthe sequence of the target nucleic acid of interest under conditionsthat permit hybridization of the detector nucleic acid to the targetnucleic acid but that exclude non-specific binding of nucleic acid(i.e., conditions are such that nucleic acid that does not have anucleotide sequence substantially complementary with the sequence of atarget nucleic acid does not bind to the target nucleic acid or to thesurface of the silicon). Optionally, the biochip can be washed to removeunbound detector nucleic acid. The hybridized nucleic acid is thendetected by any suitable detection means. For example, if the detectornucleic acid is labeled with a fluorescent molecule, the fluorescencecan be detected.

In a further embodiment, a nucleic acid complementary for a targetnucleotide sequence is contacted with a surface of a silicon biochip ofthe present invention such that the nucleic acid binds to and becomesimmobilized on the silicon. The silicon layer can be prepared so as tohave microcavities. The nucleic acid containing sample to be assayed forthe presence of one or more target nucleic acid sequences can beprovided on the silicon surface in a sol-gel composition. Optionally,the biochip can be washed to remove unbound nucleic acid. The siliconbiochip is then contacted with a nucleic acid containing sample to bescreened for the presence of the target nucleotide sequence underconditions that permit hybridization of nucleic acids comprising thetarget nucleotide sequence with the immobilized nucleic acid but thatexclude non-specific binding of nucleic acid. Optionally, the biochipcan be washed to remove unbound nucleic acid. The hybridized nucleicacid is then detected by any suitable detection means.

In one embodiment, hybridization of nucleic acids is carried out understringent hybridization conditions. As used herein, “stringent”conditions for hybridization refers to conditions wherein hybridizationis typically carried out at about 12 to 25 degrees Celsius (C) below theeffective melting temperature (Tm) of the DNA hybrid. The meltingtemperature, Tm, is described by the following formula (Beltz et al.,1983):Tm=81.5 C+16.6 Log [Na⁺]+0.41 (% G+C)−0.61 (% formamide)−600/length ofduplex in base pairs.

Washes can be carried out as follows:

(1) Once or twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS(low stringency wash); and/or

(2) Once at Tm−20 C for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

In one embodiment, a nucleic acid, for example, the detector nucleicacid, is labeled with a detectable moiety, such as a fluorescentmolecule. Examples of detectable moieties include, but are not limitedto, various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. The detectable substance may be coupled or conjugated eitherdirectly to the nucleic acid or indirectly, though an intermediate (suchas, for example, a linker known in the art) using techniques known inthe art. Examples of suitable enzymes include, but are not limited to,horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Examples of suitable prosthetic group complexesinclude, but are not limited to, streptavidin/biotin and avidin/biotin.Examples of suitable fluorescent materials include, but are not limitedto, umbelliferone, fluorescein, fluorescein isothiocyanate, CascadeBlue, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride,Texas Red, Oregon Green, cyanine (e.g., CY2, CY3, and CY5),allophycocyanine or phycoerythrin. An example of a luminescent materialincludes luminol. Examples of bioluminescent materials include, but arenot limited to, luciferase, luciferin, green fluorescent protein (GFP),enhanced GFP (Yang et al., 1996), and aequorin. Hybridization of thedetector nucleic acid is indicative of the presence of the targetnucleic acid. In one embodiment, hybridization of the detector nucleicacid to the target nucleic acid is detected using atomic forcemicroscopy (AFM). In another embodiment, hybridization of the detectornucleic acid to the target nucleic acid is detected by detecting thepresence of the detectable moiety attached to the detector nucleic acid.In an exemplified embodiment, the detectable moiety is a fluorescentmolecule.

In another embodiment, the detector nucleic acid is labeled with a firstmoiety that can bind to or be bound by a second moiety. In oneembodiment, the first moiety is digoxigenin. The digoxigenin moleculecan be incorporated into the nucleic acid molecule using digoxigeninconjugated nucleotides (e.g., digoxigenin-dUTP). The digoxigeninmolecule can be detected using an antibody that binds to digoxigeninwherein the antibody has a detectable moiety, such as a fluorescentmolecule, attached thereto. Alternatively, the antibody bound todigoxigenin can be detected by a second antibody that binds to theantidigoxigenin antibody wherein the second antibody has a detectablemoiety, such as a fluorescent molecule, attached thereto. In anotherembodiment, a biotin-avidin or biotin-streptavidin system can be used.Thus, for example, the nucleic acid can have one or more biotinconjugated nucleotides (e.g., biotin-dUTP) incorporated into it. Thebiotin moiety can be detected using avidin, streptavidin, or otherbiotin-binding molecules that have a detectable moiety, such as afluorescent molecule, attached thereto. Fluorescent moleculescontemplated within the scope of the invention include, but are notlimited to, umbelliferone, fluorescein, fluorescein isothiocyanate,Cascade Blue, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride, Texas Red, Oregon Green, cyanine (e.g., CY2, CY3, and CY5),allophycocyanine or phycoerythrin.

The present methods can be used to detect nucleic acid sequencesassociated with animals, including mammals (e.g., humans), plants,bacteria, viruses, fungi, protozoans, and the like. In one embodiment,the target nucleic acid sequence is from a Respiratory Syncytial Virus(RSV). In an exemplified embodiment, a nucleic acid (SEQ ID NO: 1)derived from RSV was immobilized on a porous silicon biochip of theinvention. The biochip was then contacted with a probe or detectornucleic acid (SEQ ID NO: 2) having a sequence complementary to theimmobilized nucleic acid (SEQ ID NO: 1) under conditions for selectivehybridization of the nucleic acids. The complete genomic sequence ofhuman RSV is known in the art (see, for example, Genbank accessionnumber NC 001781) (SEQ ID NO: 3). Any sequence within SEQ ID NO: 3, orthe complement thereof, that is of sufficient length and sequence forselective hybridization to an RSV nucleotide sequence is contemplatedfor use with the methods and materials of the present invention. Thus,all fragments and variants of the sequence shown in SEQ ID NO: 3, or thecomplementary sequence of SEQ ID NO: 3, are contemplated for use in thepresent invention.

Probes or detector nucleic acids of the invention can optionallycomprise a detectable label or reporter molecule, such as fluorescentmolecules, enzymes, radioactive moiety, and the like. Probes or detectornucleic acids of the invention can be of any suitable length for themethod or assay in which they are being employed. Typically, probes ordetector nucleic acids of the invention will be 10 to 500 or morenucleotides in length. Probes or detector nucleic acids that are 10 to20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to90, 91 to 100, or 101 or more nucleotides in length are contemplatedwithin the scope of the invention. In one embodiment, probes or detectornucleic acids are any of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and so forth up to 100nucleotides in length. Probes or detector nucleic acids of the inventioncan have complete (100%) nucleotide sequence identity with thepolynucleotide sequence, or the sequence identity can be less than 100%.For example, sequence identity between a probe or detector nucleic acidsand a sequence can be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%or any other percentage sequence identity so long as the probe ordetector nucleic acids can hybridize under stringent conditions to anucleotide sequence of a target nucleic acid.

As used herein, the terms “nucleic acid,” “polynucleotide,” and“oligonucleotide” refer to a deoxyribonucleotide, ribonucleotide, or amixed deoxyribonucleotide and ribonucleotide polymer in either single-or double-stranded form, and unless otherwise limited, would encompassknown analogs of natural nucleotides that can function in a similarmanner as naturally-occurring nucleotides. Polynucleotide sequencesinclude the DNA strand sequence that is transcribed into RNA and the RNAstrand that is translated into protein. The complementary sequence ofany nucleic acid, polynucleotide, or oligonucleotide of the presentinvention is also contemplated within the scope of the invention.Polynucleotide sequences also include both full-length sequences as wellas shorter sequences derived from the full-length sequences.

The subject invention also concerns variants of the polynucleotides ofthe present invention, including variants of the RSV sequence shown inSEQ ID NO: 3. Variant sequences include those sequences wherein one ormore nucleotides of the sequence have been substituted, deleted, and/orinserted. The nucleotides that can be substituted for naturalnucleotides of DNA have a base moiety that can include, but is notlimited to, inosine, 5-fluorouracil, 5-bromouracil, hypoxanthine,1-methylguanine, 5-methylcytosine, and tritylated bases. The sugarmoiety of the nucleotide in a sequence can also be modified andincludes, but is not limited to, arabinose, xylulose, and hexose. Inaddition, the adenine, cytosine, guanine, thymine, and uracil bases ofthe nucleotides can be modified with acetyl, methyl, and/or thio groups.Sequences containing nucleotide substitutions, deletions, and/orinsertions can be prepared and tested using standard techniques known inthe art.

Polynucleotides contemplated within the scope of the subject inventioncan also be defined in terms of more particular identity and/orsimilarity ranges with those sequences of the invention specificallyexemplified herein. The sequence identity will typically be greater than60%, preferably greater than 75%, more preferably greater than 80%, evenmore preferably greater than 90%, and can be greater than 95%. Theidentity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequenceexemplified herein. Unless otherwise specified, as used herein percentsequence identity and/or similarity of two sequences can be determinedusing the algorithm of Karlin and Altschul (1990), modified as in Karlinand Altschul (1993). Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (1990). BLAST searches can beperformed with the NBLAST program, score=100, wordlength=12, to obtainsequences with the desired percent sequence identity. To obtain gappedalignments for comparison purposes, Gapped BLAST can be used asdescribed in Altschul et al. (1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) can be used. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide moleculeshaving sequences which are sufficiently homologous with thepolynucleotide sequences exemplified herein so as to permithybridization with that sequence under standard stringent conditions andstandard methods (Maniatis et al., 1982).

Biochips of the invention have many advantages, including highresponsiveness and selectivity, and are inexpensive. Two primaryadvantages make nanoscale porous silicon based DNA biochips a veryattractive option: (i) enormous surface area, which ranges from about 90to 783 m²/cm³, and which provide numerous sites for potential species toattach and (ii) its room temperature luminescence spans the visiblespectrum which makes it an effective transducer. In one embodiment,binding DNA to porous silicon involves coating sol-gel materialcontaining DNA on an oxidized silicon surface.Tetra-ethyl-ortho-silicate (TEOS) can be used to provide a stablecoupling between two non-bonding surfaces: an inorganic surface to abio-molecule (DNA). The most interesting feature of porous silicon isits room temperature visible luminescence. Porous silicon microcavityresonators possess the unique characteristics of line narrowing andluminescence enhancement (Canham, 1990). The emission peak position iscompletely tunable by modifying the coating over the surface of poroussilicon (Lauerhans et al., 1993). The present invention demonstrates theoptoelectronics properties of and the compatibility of the poroussilicon fabrication process with the usual silicon technology. Further,a mechanical non-fluorescence based approach using AFM technique todetect DNA hybridization can be used with the present invention. Inanother embodiment, hybridization of sDNA with complementary DNA (cDNA)having a fluorescent probe molecule attached to the cDNA is utilized.Hybridization on the DNA biochip can be detected using photoluminescencetechnique. Results using a DNA biochip of the present invention anddetection techniques are summarized in Tables 1-2. The present inventionwas found to be more sensitive, economical and time efficient thanexisting technologies as shown in Table 2.

Any element of any embodiment disclosed herein can be combined with anyother element or embodiment disclosed herein as if the combination isexplicitly disclosed or exemplified herein, and such combination iscontemplated within the scope of the present invention.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Materials and Methods

Compositions and Reagents.

A crystalline n-type silicon wafer with resistivity ranging between 0.4and 0.6 Ωcm was used for preparing porous silicon (PS) layers by dippingin a solution of hydrogen fluoride (HF) and ethanol. The target nucleicacid having DNA sequence3′-GATCCTCGGTAACACAGTACGATACCGTTTTGATTTACATGTCGTAGGTTATTTTTAGCACCTTAGTATTTCTGTAAAAGATTGCCCACGCTAATA-5′(SEQ ID NO: 1) and tetra-ethyl-ortho-silicate (TEOS), HCl, and HNO₃ werecontacted with a porous silicon surface (prepared as described herein)for a sufficient period of time to permit the nucleic acid to bindthereto. Then, a detector nucleic acid labeled with a fluorescentmolecule and having the complementary RSV F genome sequence5′-CTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAATGTACAGCATCCAATAAAAATCGTGGAATCATAAAGACATTTTCTAACGGGTGCGATTAT-3′(SEQ ID NO: 2) was used for hybridization.

Preparation of Micro Cavities on Silicon Wafer.

With reference to FIG. 1, anodic etching was used to prepare poroussilicon wafers using an electrolyte solution 12 containing 49% highpurity aqueous HF and 50% ethanol. A 14.4 cm² exposed area of thepolished, crystalline n-type silicon wafer 18 with resistivity rangingbetween 0.4 and 0.6 Ωcm was etched for 5 minutes in a Teflon cell 10 ata constant anodic current of 40.3 mA/cm². The cell 10 contains an O-ring14.

A 200 nm gold layer was deposited by sputtering at the bottom of thesilicon wafer 18 to ensure ohmic contact. The cathodic contact was madeusing a platinum mesh 16 that is in contact with the solution. After theetching process was achieved the wafer was rinsed in ethanol then blowndry in a nitrogen environment. The advantage of this cell geometry isthe simplicity of equipment as shown in FIG. 1. The presence of adifference in the potential between the top and the bottom electrodes ofsuch a cell, leads to different values of the local current density(Jarimaviciute et al., 2003).

Procedure of Immobilization of sDNA onto Porous Silicon.

The method used for binding DNA to the silicon involves coating theoxidized surface of porous silicon with a sol-gel containing singlestranded DNA. Sol-gel is a colloidal suspension of silica particles thatis gelled to form a solid. The resulting porous gel can be chemicallypurified and consolidated at high temperatures into high purity silica.The idea behind the sol-gel optical sensors is based on changes ofoptical parameters of active (sensing) molecules (DNA) physicallyentrapped in sol-gel thin films. Those changes are induced by changingexternal physico-chemical parameters such as temperature, hydrostaticpressure or presence of analyte molecules. There are several kinds ofoptical signals which could be used as analytical response of suchsensors, for instance: intensity of light absorbed or emitted by thesensing molecules, time of luminescence decay (Chan, et al., 2000).Non-labeled DNA comprising the nucleotide sequence3′-GATCCTCGGTAACACAGTACGATACCGTTTTGATTTACATGTCGTAGGTTATTTTTAGCACCTTAGTATTTCTGTAAAAGATTGCCCACGCTAATA-5′(SEQ ID NO: 1) was immobilized using tetraethylorthosilicate (TEOS)spread over the surface of the silicon wafer to immobilize DNA in themicrocavities. A mixture of 25 μL of TEOS, 5 μL of 0.1 M HCl and 204, ofde-ionized water (DI) were mixed in a vial (solution A). The last stepinvolved mixing 2 μL single stranded DNA (sDNA) stock solutioncontaining the oligonucleotide (SEQ ID NO: 1) and 3 μL DI water in 5 μLof solution A, resulting in a dilution of solution A to 50%. The pH wascontrolled near 7 during the mixing procedure described above. Thesingle stranded DNA stock solution contains (1 mg) DNA in 1 mL DI water.

A schematic diagram is shown in FIGS. 2A-2C. FIG. 2A shows the procedurefor immobilizing the sDNA on the porous silicon using TEOS. FIG. 2Bshows the immobilized sDNA on porous silicon and FIG. 2C shows thehybridization of cDNA that corresponds to RSV F genome having thenucleotide sequence5′-CTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAATGTACAGCATCCAATAAAAATCGTGGAATCATAAAGACATTTTCTAACGGGTGCGATTAT-3′(SEQ ID NO: 2). This complementary strand was labeled with a fluorescentmolecule.

AFM Characterization of DNA Biochip.

There are various modes of atomic force microscope (AFM) operation, themost common are: Non-contact mode, contact mode, and tapping mode.Tapping mode is the preferred mode of operation in the case of thisstudy since it has features that allow better quality imaging withlittle deleterious effects on the sample. Analysis of samples in suchmode provides higher lateral resolutions, which is critical whenanalyzing DNA immobilized over a silicon surface. Lower forces and lessdamage to soft samples make it suitable for DNA structural analysis andsample scraping is virtually eliminated since there are minimal or nolateral forces exerted on the sample. General features of thesemolecules pertinent to important biological processes are now beingcharacterized using this technique. AFM software is used to obtainquantitative, three-dimensional images of surfaces with ultra-highresolution. AFM provides measurements of surface roughness, grain size,and grain size distribution. All analyses were conducted in air and thesamples were brought to room temperature before AFM analysis.

Optical Microscopic Studies of DNA Biochip.

Optical microscopic pictures were recorded for porous silicon wafers andporous silicon containing sDNA and after hybridization of sDNA withcDNA. (FIGS. 4A-4C). The optical microscope of sDNA attached modifiedsingle-stranded oligonucleotides were recorded using a Vanox researchgrade optical microscope for homogeneous hybridization studies. Thetransverse mode profile for the disk and evanescent field used forsensing is equivalent to that of a slab waveguide with the samethickness and refractive indices. Therefore, one can take advantage ofenhanced power at the surface of the porous silicon containingmicrocavities, having the same penetration depth and relative claddingpower as in the straight waveguide structure.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Preparation of Porous Silicon Wafer with Immobilized DNA

Porous silicon provides numerous sites for nucleic acid sequence toattach. The porous layer of silicon was fabricated by means of theelectrochemical etching in HF solution. FIGS. 4A-4C show the surface ofporous silicon after etching. In this case, only a small quantity ofcharge was generated to generate the pores over the silicon wafersurface. Single strand DNA (sDNA) (SEQ ID NO: 1) of RSV virus wasimmobilized on PS. A fluorescent probe molecule was attached to a cDNA(SEQ ID NO: 2) having a sequence complementary to the sequence in SEQ IDNO: 1 and then brought into contact with the PS having the sDNAimmobilized thereon under conditions sufficient for hybridization of thecDNA to the immobilized sDNA. The fluorescent molecule on the cDNAprovides the means of detecting the extent of hybridization of the cDNAto the sDNA.

EXAMPLE 2 SEM Characterization of Microcavities on Silicon Wafer

A cross-section SEM picture of a porous silicon microcavity is shown inFIG. 3. This picture was taken transversally to the silicon surface andillustrates hemispherical structures over the entire surface. Thesestructures represent the beginning stage of porous silicon formation.Furthermore, pore depths varying between 0.5 and 0.75 μm are highlightedin the figure by white-dotted lines.

EXAMPLE 3 Optical Microscopy (Epi) Studies of DNA Biochip

Epi indicates incident illumination and has been used in the presentinvention. The reflection and refraction of light according to themultiple wavelet concepts, now known as the Huygens' principle. When thewavefront encounters the interface between the two media, a portion ofthe light is reflected and another part is refracted. The periodic rowsof miniature semicircular red waves represent the Huygens wavelets thattogether compose the incident and reflected wave fronts. Wavelets thatpenetrate the media boundary to become refracted are portrayed in blue,as is the line passing through the center of the refracted beam thatdenotes their direction of propagation. According to the Huygens modelof light, a small portion of each angled wavefront impacts the secondmedium before the rest of the front reaches the interface. This portionof the wavefront begins to move through the second medium while the restof the wave is still traveling in the first medium. The speed at whichthe wavelets travel through is dependent on the refractive indices ofthe media. If the second medium has a higher refractive index than thefirst, then the light slows down, and vice versa. Since in either casethe wavefront is then traveling at two different speeds, it bends intothe second medium, thus changing the angle of propagation. The mostcommon oil-immersion objective in use in routine microscopy is used formagnification of ×100. Fluorescence is a process where a substance afterhaving absorbed light (photons) emits a radiation the wavelength(colour) of which is longer than that of the absorbed light, and wherethis emission stops immediately after cessation of the excitation. Thisphenomenon is used to understand the DNA hybridization in DNA biochip.Besides the “classical” excitation of fluorescence in a light microscopeit is possible today to obtain the same emission effect. Fluorescenceoccurs either as autofluorescence of biological and/or inorganicstructures or as so called secondary fluorescence after a treatment ofthe specimen with special dyes (e.g., fluorochromes, fluorescentmarkers). The microcavity design has an advantage over the single layerstructure as the refractive index of the surrounding material increasesthe reflectivity spectrum to shift. A blue shift is predicted becausethe pores are filled with sol-gel material with different refractiveindex as shown in FIGS. 4A-4C. This is further demonstrated during theoptical microscopy studies. A optical microscope was used to achievefluorescence-aided molecule sorting (FAMS) and enabled simultaneousanalysis of DNA interactions at the level of a single strand. This wasperformed by labeling cDNA (SEQ ID NO: 1) corresponding to RSV F genomicsequence. The cDNA probe comprised the nucleotide sequence5′-CTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAATGTACAGCATCCAATAAAAATCGTGGAATCATAAAGACATTTTCTAACGGGTGCGATTAT-3′(SEQ ID NO: 2) and was used for hybridization. This complementary strandwas labeled with a fluorescent molecule that serves as donor-acceptorpair for a Forster resonance energy transfer. FAMS permits equilibriumand kinetic analysis of macromolecule-ligand interactions; this wasvalidated by measuring with sDNA and cDNA. FAMS is a general platformfor ratio metric measurements that report on structure, dynamics,stoichiometries, environment, and interactions of diffusing orimmobilized molecules, thus enabling detailed mechanistic studies andultra sensitive diagnostics (Garcia-Parajo et al., 2001).

EXAMPLE 4 UV Studies of DNA Biochip

UV-spectra have shown the retention of the fluorophore in the modifiedcDNA. The absorbance at 333-340 nm and at 260 nm due to fluorophore andDNA, respectively, and fluorescence emission spectra at 500-520 nmwavelengths clearly confirmed the retention of the chromophore in theoligonucleotides. The relative enhancement in the intensity of peak isdue to the fluorescence molecule attached to cDNA. A fluorophore layerplaced on top of porous silicon will experience an enhancement of theinput optical signal. The effect of field enhancement in microcavitiescan be interpreted as an increase of absorption efficiency of thefluorophore due to increased interaction length of the incident fieldwith an absorbing molecule. Therefore, an increase in amount offluorescent photons generated from the molecule at the microcavitiesversus the linear waveguide is proportional to a number of fluorescencemolecule or hybridization with cDNA. Therefore, the advantage of themicrocavity format versus waveguide format for analytical applicationsis the amount of fluorescence molecules present at surface of poroussilicon or hybridization. Therefore, the fluorescence signal from themolecules near the microcavity is increased

EXAMPLE 5 AFM Studies of DNA Biochip-AFM Surface Analysis of DNAImmobilized on Microporous Cavity

Surface images of non-hybridized sDNA on PS and sDNA hybridized with anRSV F specific oligonucleotide cDNA probe were taken using a DigitalInstruments Atomic Force Microscope (AFM) equipped with nanoscopedimension 3000 software. FIGS. 7A and 7B show a two dimensional pictureof a section of the microporous silicon wafer with a single strand DNAbundle attached to a cavity. FIGS. 7A and 7B show a “horse shoe” likestructure coming out of the microcavity.

FIG. 8A confirms the sDNA bundle shape and provides a better image ofthe surface profile of the sol gel/sDNA mixture. Further 3D AFM analysisof this image provides more information about the dimensions and theform of the ssDNA bundle, as shown in FIG. 8A. In FIG. 8B notice thatthis value is at least twice as shallow as the value determined by SEM.This is due to the application of the sol gel film which has partiallyfilled the microcavities.

EXAMPLE 6 Photoluminescence Studies of RSV DNA before and afterHybridization

Photoluminescence (PL) was used to study the effectiveness with whichthe fluorescently tagged RSV oligonucleotide probe molecules hybridizeto the fixed sDNA molecules on the surface of the porous silicon. Foursamples were selected for this study: two (samples “a” and “b”) withsDNA only immobilized on the surface and two hybridized samples havingthe oligonucleotide probe hybridized to the sDNA (samples “c” and “d”).All samples were illuminated with a helium cadmium (He Cd) laser at 325nm and 55 mW. The laser beam was kept at 1.5 mm in diameter to minimizethe damage to the DNA molecules. FIG. 9 shows the PL spectra of sDNAfixed on two different “sDNA only” samples (a, b), and two hybridizedDNA samples (c, d). A clear increase in the PL intensity was observedafter hybridization of the single strand DNA with the RSV cDNA.

FIG. 9 shows that all peaks are found around 505 nm with minor shiftingin the wave length in the order of 5 to 10 nm between the sDNA samplesand the hybridized ones. However a significant change in the intensitywas clearly perceived between sDNA and hybridized DNA samples. WhilesDNA only samples (a, b) did not show any significant peak, thehybridized (c, d) samples did show two peaks. The smaller peak wasregistered at 382 nm which corresponds to the color blue. The peak withhigher intensity corresponds to the green color with a wavelength of 508nm. This clearly demonstrates a noticeable change that could be used toquantify the extent of hybridization on the surface. Furthermore, the PLspectra are in concordance with the images obtained by fluorescentmicroscopy, where bright blue and green areas were observed on thesurface of the PS having the cDNA hybridized thereon.

TABLE 1 Fluorescence and optical microscopic studies of DNA biochip.Hybridized Technique sDNA (sDNA: cDNA) Optical Dark green color wasBright blue and green Microscopy observed with very little fluorescenceobserved fluorescence observed Mass difference “Horse shoe” like sDNAHybridized DNA structure (AFM) bundles were found with twice the sDNAimages were observed Photoluminescence No significant Relatively highintensity Studies (PL) photoluminescence was spectra with blue peakobserved (382 nm) and green peak (508 nm)

TABLE 2 Comparison of existing RSV detection techniques with DNA biochipof present invention. Time of Techniques detection SelectivitySensitivity Reference Radio- Days Likely to be  79% Meurman immunoassaypositive et al., 1984a Immuno- Days Lower background  75% Walliverfluorescence absorbance can be et al., (Serology) obtained if the 1980RSV antigen is partially purified CF Assays Days 5-6% sensitivity  25%Richardson et al., 1978 ELISA Days 88% selectivity  92% Meurman, et al.1984b DNA Biochip <Minutes Highly selective 100% Present work

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

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We claim:
 1. A silicon-based biochip, wherein said biochip comprises asilicon wafer comprising a bottom layer that comprises gold and providesohmic contact and said biochip further comprises microcavities in saidsilicon wafer, and wherein a nucleic acid is immobilized on the surfaceof said silicon wafer and is provided in a sol-gel composition.
 2. Thebiochip according to claim 1, wherein said nucleic acid is in singlestranded form.
 3. The biochip according to claim 1, wherein said biochipis prepared by exposing the surface of a silicon wafer to a solution ofhydrofluoric acid (HF) and ethanol and a constant anodic current.
 4. Thebiochip according to claim 1, wherein said microcavities have a depth offrom about 0.5 μm to about 0.75 μm.
 5. The biochip according to claim 1,wherein said nucleic acid is a Respiratory Syncytial Virus (RSV) nucleicacid.
 6. The biochip according to claim 5, wherein said RSV nucleic acidcomprises the nucleotide sequence shown in SEQ ID NO:1.
 7. The biochipaccording to claim 5, wherein said RSV nucleic acid comprises thenucleotide sequence shown in SEQ ID NO:3, or the complement thereof, orsaid RSV nucleic acid is a fragment of SEQ ID NO:3, or the complementthereof, that is of sufficient length for selective hybridization to anRSV nucleotide sequence.
 8. The biochip according to claim 1, whereinsaid silicon wafer is a crystalline n-type silicon wafer.
 9. The biochipaccording to claim 1, wherein said sol-gel composition comprisestetra-ethyl-ortho-silicate (TEOS).
 10. The biochip according to claim 1,wherein said bottom layer is 200 nm in thickness.